Keratins are the most abundant proteins in epithelial cells, in which they occur as a cytoplasmic network of 10-12 nm wide intermediate filaments (IFs). They are encoded by an evolutionarily conserved multigene family, with > 49 individual members subdivided into two major types. The pairwise regulation of type I and type II keratin genes reflects a heteropolymerization requirement shared by all keratin IFs. Most keratin genes are regulated in pairwise, epithelial tissue-type and differentiation-specific fashion, the functional basis of which remains ill understood. A major role fulfilled by keratin IFs is to act as a resilient yet pliable scaffold that endows epithelial cells with the ability to sustain mechanical and non-mechanical stresses. Inherited mutations affecting the coding sequence of keratins are responsible for a large number of epithelial fragility disorders. Additional functions, manifested in a sequence- and context-dependent fashion, have been identified in recent years. Here we seek to further our understanding of the properties and functions of type II keratins expressed in complex epithelial tissues, including K1, K5 and K6 isoforms, and test our model that differential keratin expression leads to distinct mechanical attributes for keratinocytes. First, we will test the ability of K1 to rescue the striking epithelial defects that occur in K6 isoform null mice. We will concentrate on the epithelial fragility in the oral mucosa and the altered wound epithelialization phenotype seen in these mice. Second, we will investigate the biochemical and genetic bases for the strain-dependency of the enhanced migration exhibited by K6 isoform null keratinocytes in an ex vivo model of wound epithelialization. Third, we will assess the specific contribution of keratin IFs to the viscoelastic properties of live epithelial cells. Fourth, we will test the notion that the self-induced ability of keratin IFs to undergo bundle formation is required for their scaffolding function in vivo. In particular, we will focus on the contribution of nonhelical C-terminal tail domains to filament bundling and function. The proposed research will provide insight into keratin filament organization and function, the significance of differential keratin expression, and the mechanisms underlying blister formation in keratin-based blistering diseases.